Project Summary/Abstract Microglia are strongly implicated in the pathogenesis of Alzheimer's disease (AD) and recent genetic studies have identified several microglial-enriched genes that influence AD risk. To study the role of these genes in AD our lab recently developed a fully-defined approach to differentiate induced pluripotent stem cells (iPSCs) into microglia. However, reprogramming erases many of the key signatures of aging, making it difficult to study the interactions between AD genes, pathology, and aging with iPSC-derived cells. One recent study developed an innovative approach to address this challenge by using Progerin, a protein associated with a premature aging disorder. However, the Progerin gene, LMNA, is not normally expressed in human neurons or glia. In contrast, another form of Progeria, Cockayne Syndrome (CS), which is caused by mutations that impair DNA repair, leads to significant neurological defects. Furthermore, two of the genes that are mutated in CS, ERCC1 and ERCC5, are highly expressed in human microglia and their deletion in mice mimics key aspects of microglial aging. Given these findings, we propose to test the hypothesis that deletion or mutations of ERCC1 and ERCC5 will produce changes in iPSC-derived microglia that mimic the effects of chronological aging and impair the response of microglia to AD-associated insults. To achieve these goals, we have assembled a multidisciplinary team who bring expertise in AD iPSC modeling, CRISPR-mediated gene deletion, and microglial differentiation and analysis, RNA-sequencing and bioinformatics, and brain organoid culture systems to test following three specific aims and hypotheses: Aim 1: Examine the impact of ERCC1/5 deletions and mutations on iPSC-derived microglial function. We hypothesize that ERCC knockout and mutant microglia will exhibit an age-associated `primed' activation state with impaired homestatic activity but exacerbated inflammatory responses. Aim 2: Do ERCC1/5 deletions and mutations model the transcriptional effects of aging observed in human brain-derived microglia? We hypothesize that deletion of these genes will produce microglia that exhibit many of the transcriptional changes associated with natural brain aging. Aim 3: Defining the intrinsic versus extrinsic effects of ERCC deletion on microglial aging with brain organoid cultures. We hypothesis that both intrinsic and extrinsic signals influence microglial aging.